PROJECT SUMMARY The goal of this proposal is to test if a discovery we recently made on glycosphingolipid trafficking in epithelial cells can be translated to clinical application as a platform for drug delivery of biologics. Mucosal surfaces represent vast areas where host tissues are separated from the environment only by a delicate but highly effective single layer of columnar epithelial cells, joined by tight junctions that are impermeable to proteins and even small peptides. So far, the lack of rational and efficient methods to circumvent this barrier has prevented the application of most therapeutic proteins and peptides for mucosal drug delivery. Endothelial cells also form vast and highly restrictive single cell thick barriers that separate most tissues from the blood stream. Most healthy non-inflamed endothelial barriers strongly limit the permeability of large molecules; thus preventing access of many protein-based biologics to cells of many tissues - even when the therapeutic proteins are applied intravenously. Here, we address these problems by testing whether non-native ?short-chain? GM1 glycosphingolipids can serve as molecular carriers for drug delivery of peptide and protein biologics. Substantial progress was made since the original submission of this proposal in 2014. Structure-function studies on transepithelial transport of the ceramide domain identified non-native GM1-species that have the combined features of enhanced uptake, transcytosis across epithelial barriers, and efficient release from cell membranes after transport. These GM1 species are studied as vehicles for cargo transport. In Aim 1, we will conclude and expand our preliminary studies testing for GM1-mediated transport of peptide and protein cargoes across mouse epithelial and endothelial barriers in vivo. The GM1 lipids are fused via their extracellular oligosaccharide domain to reporter peptides yielding robust signals for tracking the molecules by imaging and biochemically. We will test for transport of larger cargoes, and for proof of principle by mucosal administration of the non-native GM1 species fused to the incretin hormone GLP-1, as model for treatment of Type II Diabetes. Transport of peptides across tight endothelial barriers (heart and brain) will be tested in vivo. Aim 2 will test for mechanism of transcytosis for the non-native GM1 species. The major hypotheses for sorting by molecular shape or by association with membrane microdomains will be examined. Aim 3 will test if the extracellular oligosaccharide domain of GM1 can be truncated or eliminated while still maintaining functionality of the GM1 ceramide domain in trafficking. We will explore novel structures of the linker peptide to replace functionalities of the oligosaccharide head groups if necessary. In Aim 4, we will test if the linker between GM1 and peptide can be designed to release the cargo after or during transcytosis. We will test if the incorporation of a cleavable ester bond, or a motif for the endosomal- protease furin, can achieve this goal.